The retinoid visual cycle refers to a multi-step pathway to recycle 11-cis retinal, a light sensitive chromophore for rod and cone visual pigments in vertebrates. A key step in the visual cycle is the conversion of all-trans retinyl ester to 11-cis retinol, which is catalyzed by an enzyme, namely isomerohydrolase. RPE65 is a membrane-associated protein predominantly expressed in the retinal pigment epithelium (RPE). RPE65 mutations are known to cause inherited retinal dystrophies. Previously, three groups including us have independently shown that RPE65 has the isomerohydrolase activity. Further, we have recently demonstrated robust isomerohydrolase activity in purified RPE65 protein reconstituted into liposomes, which provides conclusive evidence indicating that RPE65 is the isomerohydrolase in the visual cycle. It has become evident that cone-dominant species have a more efficient retinoid visual cycle than rod-dominant species. We have reported recently that RPE65 from cone-dominant chicken has substantially higher specific isomerohydrolase activity, compared to mammalian RPE65. In addition, several lines of evidence suggest that there exists another isomerase alternative to RPE65 in cone-dominant retinas. A recent study has shown that there is an RPE65-independent isomerohydrolase activity in cone-dominant zebra fish retina. However, the enzyme responsible for the RPE65-independent isomerohydrolase activity has not been previously identified. In preliminary studies, we have identified, cloned and expressed two novel RPE65 homologues, namely zebra fish RPE65c (zRPE65c) and 13-cis isomerohydrolase (13cIMH) from cone-dominant zebra fish, which share 78% and 77% amino acid identities, respectively, to zebra fish RPE65 (zRPE65 or zRPE65A). RT-PCR showed that zRPE65 and zRPE65c are both expressed in the eye. In isomerohydrolase assay, zRPE65c generated both 11-cis retinol and 13-cis retinol, while zRPE65 generated exclusively 11-cis retinol from the same substrate, all-trans retinyl ester. This result suggests that zRPE65c may be responsible for the RPE65-independent isomerohydrolase activity in the zebra fish retina. Interestingly, 13cIMH is expressed in the brain but not in the eye. In the same isomerohydrolase assay, 13cIMH generated exclusively 13-cis retinol from all-trans retinyl ester, suggesting that it may play a role in generating 13-cis retinoic acid, which has a critical role in the regulation of development and neurological functions. Based on these findings, we hypothesize that the more efficient visual cycle in cone-dominant species can be ascribed to the higher isomerohydrolase activity of RPE65 or/and expression of other isomerohydrolases in the retina. In this project, we propose the following studies to elucidate molecular mechanism underlying the efficient visual cycle in cone-dominant species. 1) To investigate the structural basis underlying the higher isomerohydrolase activity in chicken RPE65. We will substitute selected residues in human RPE65 by their chicken counterparts, to generate a human RPE65 mutant with an isomerohydrolase activity as high as chicken RPE65. 2) To characterize zRPE65c and establish its role in the RPE65-independent visual cycle in cone-dominant species. Previous evidence suggested that the alternative isomerase is likely to exist in retinal Muller cells in the cone-dominant retinas. This study will identify cellular localization of zRPE65c in the zebra fish retina. The kinetics and characteristics of its enzymatic activity will be measured and compared with RPE65. We will also knockdown zRPE65 and zRPE65c individually, and knockdown both by morpholino approach, to evaluate the effect of the knockdown on isomerohydrolase activity, 11-cis retinoid content in the eyecups and rod and cone structure and function. 3) To identify key residues responsible for different products generated by RPE65 and 13cIMH. 13cIMH is the first identified enzyme which generates 13-cis retinoid. We will knockdown the expression of 13cIMH and determine if it affects the generation of 13-cis retinoid acid in the brain. We will compare the sequences of zRPE65 and 13cIMH, and generate mutations to identify key residues determining the products specificity of the isomerohydrolase, i.e., RPE65 generates 11-cis retinol while 13cIMH generates only 13-cis retinol. These studies will not only identify the alternative isomerohydrolase in cone-dominant retina, but also contribute to the understanding of mechanism for the isomerization/hydrolysis reaction catalyzed by RPE65.